High Frequency of Prostate Antigen-Directed TCells in Cancer Patients Compared to Healthy
Age-Matched Individuals
Ole Forsberg,
1Bjo¨rn Carlsson,
1Per-Uno Malmstro¨m,
2Gustav Ullenhag,
3Thomas H. To¨tterman,
1and Magnus Essand
1*
1
Clinical Immunology Division, Rudbeck Laboratory,Uppsala University,Uppsala, Sweden
2
Experimental Urology Division,Uppsala University Hospital,Uppsala, Sweden
3
Oncology Division,Uppsala University Hospital,Uppsala, Sweden
BACKGROUND. In order to obtain a sustained cytotoxic T lymphocyte (CTL) response against cancer cells it is preferable to have CTLs directed against multiple peptide epitopes from numerous tumor-associated antigens.
METHODS. We used a Flow Cytometry-based interferon (IFN)-g secretion assay with peptide-pulsed C1R-A2 as antigen-presenting cells to analyze whether CD8þT cells directed against any of 24 HLA-A*0201-binding peptides from 15 prostate-associated proteins can be found in the peripheral blood of patients with localized prostate cancer. We also investigated whether multiple prostate antigen-specific CD8þT cells can be generated simultaneously, from a naı¨ve T cell repertoire. In that case, dendritic cells (DCs) from peripheral blood of healthy donors were divided in six portions and separately pulsed with six peptides. The peptide-pulsed DCs were then pooled and used to stimulate autologous T cells. The T cells were re-stimulated with peptide-pulsed monocytes.
RESULTS. We found prostate antigen-restricted CD8þT cells in the peripheral blood in 48 out of 184 (26.1%) analyzed samples from 25 cancer patients. This is significantly higher than 17 out of 214 analyzed samples (7.9%) from 10 healthy age-matched male individuals (P ¼ 0.0249). In the cases when antigen-specific T cells could not be detected, we were able to generate IFN-g- producing CD8þT cells specific for up to three prostate antigens simultaneously from a naı¨ve T cell repertoire.
CONCLUSIONS. CD8þT cells directed against prostate antigen peptides can be found in, or generated from, peripheral blood. This indicates that such T cells could be expanded ex vivo for adoptive transfer to prostate cancer patients. Prostate 69: 70–81, 2009. #2008 Wiley-Liss, Inc.
KEY WORDS: prostate cancer; T cell; dendritic cell; HLA-A2 peptides
INTRODUCTION
Prostate cancer is a leading cause of cancer-related death among men in Western countries [1]. For organ- confined prostate cancer radical prostatectomy is generally implemented. It has a fairly high cure rate.
However, a significant proportion of patients relapse and for these patients curative treatments are not available. One therapeutic strategy that has evolved over the last two decades is cancer vaccines that aim at generating a potent and long lasting anti-tumor T cell response. Initial attempts were made with tumor cell
Ole Forsberg and Bjo¨rn Carlsson contributed equally to this work.
Grant sponsor: Swedish Cancer Society; Grant number: 4419-B06- 07XCC; Grant sponsor: Gunnar Nilsson’s Cancer Foundation; Grant number: E35/07; Grant sponsor: Lion’s Cancer Foundation in Uppsala.
*Correspondence to: Magnus Essand, Clinical Immunology Divi- sion, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
E-mail: magnus.essand@klinimm.uu.se
Received 28 May 2008; Accepted 21 August 2008 DOI 10.1002/pros.20858
Published online 22 September 2008 in Wiley InterScience (www.interscience.wiley.com).
lysate or irradiated tumor cells combined with an adjuvant or with tumor cells genetically modified to secrete cytokines [2]. Vaccination with tumor- associated antigens (TAAs) has also been applied [3]
and TAAs have been combined with ex vivo-cultured dendritic cells (DCs) as a DC-based vaccine in order to optimize the activation of naı¨ve T cells [4,5]. Further- more, ex vivo activation and expansion of TAA- directed T cells followed by adoptive transfer of such T cells has also been utilized [6,7].
Prostate cancer is well suited for adoptive T cell therapy since the prostate gland is an organ not essential for life. Therefore, all tissue-specific proteins can be exploited as potential T cell targets. Today, more than thirty genes have been reported to be specifically or preferentially expressed by prostate cells. They are listed in a recent review [8]. So far the PSA, PSMA, PSCA, PAP, Kallikrein-4, TARP, Prostein, Trp-p8, STEAP, and PAGE-4 proteins have been evaluated as T cell antigen targets [9–27]. In general, these proteins were screened for peptides with ability to bind HLA- A*0201 and generate specific cytolytic T lymphocytes (CTLs) ex vivo.
In order to obtain a broad and long-lasting CTL response it may be preferable to use a panel of TAAs for T cell stimulation. Therefore, in this article we characterize the binding property of 24 HLA-A*0201- restricted peptides derived from 15 proteins that are specifically or preferentially expressed by normal prostate and prostate cancer cells. We report that circulating peptide-specific CD8þ T cells are often found in the peripheral blood of prostate cancer patients with localized disease and that there is a significant difference in T cell occurrence in cancer patients compared to age-matched healthy individuals.
For patients in whom circulating prostate antigen- directed T cells cannot be detected we describe an ex vivo protocol to generate CD8þT cells against multiple peptides simultaneously.
MATERIALS AND METHODS Cell Lines
The human EBV-transformed B lymphoblastoid cell line C1R [28] does not express endogenous HLA-A or -B molecules [29]. C1R-A2 cells express a transfected genomic clone of HLA-A2.1 [30]. We obtained C1R-A2 from Dr. J. Berzofsky, Vaccine Branch, National Cancer Institute, Bethesda, MD. C1R-A2 was cultured in RPMI-1640 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 100 U/
ml of penicillin and 100 mg/ml of streptomycin (1% PEST) (Invitrogen), and 200 mg/ml of geneticin (Sigma Chemicals, St. Louis, MO) to ensure HLA-A2.1 transgene expression. The T2 cell line is deficient in
transporters associated with antigen processing (TAP) and therefore unable to present endogenous peptides through the proteasome degradation pathway [31]. T2 was cultured in RPMI-1640 supplemented with 10%
FBS and 1% PEST.
Flow Cytometry Analysis
C1R-A2 and mature monocyte-derived DCs were stained for cell surface expression of HLA-ABC, HLA- DR, CCR7, CD1a, CD19, CD20, CD40, CD54, CD80, CD83, and CD86 by using specific fluorophore-labeled antibodies (BD Biosciences, San Diego, CA) and isotype-relevant fluorophore-labeled negative control antibodies (BD Biosciences). Furthermore, cells were incubated for 30 min at 48C with the mouse monoclonal anti-HLA-A2.1 antibody BB7.2 (a kind gift from Dr. J Berzofsky, Vaccine Branch, National Cancer Institute).
After washing, cells were stained with FITC-labeled rabbit anti-mouse antibody (DAKO, Copenhagen, Denmark). Cells were washed and analyzed by Flow Cytometry on a FACSCaliburTM(BD Biosciences).
Peptide Epitope Selection, Peptide Synthesis and Peptide Binding
Proteins reported to be specifically or preferentially expressed in prostate and prostate cancer cells were analyzed for putative HLA-A*0201-binding peptides using the online computer algorithms BIMAS [32]
(bimas.cit.nih.gov/molbio/hla_bind/) and SYFPEI- THI [33] (www.syfpeithi.de). Thirty-one proteins, listed in a recent review [8], were analyzed and peptides from 15 proteins with high specificity for prostate cells were selected: prostate-specific antigen (PSA) [34], prostatic acid phosphatase (PAP) [35], prostate-specific membrane antigen (PSMA) [36], prostate-specific transglutaminase (TGM4) [37], pros- tate stem cell antigen (PSCA) [38], T cell receptor g-chain alternate reading frame protein (TARP) [39,40], six transmembrane epithelial antigen prostate (STEAP) [41], prostase/human kallikrein-4 (hKLK4) [42], prostate-specific gene with homology to a G protein- coupled receptor (PSGR) [43], Trp-p8 [44], prostate and testis expression (PATE) [45], prostate, ovary testis and placenta expression (POTE) [46], six transmembrane protein of prostate 1 (STAMP1) [47], androgen-induced bZIP (AIbZIP) [48] and novel prostate-specific antigen (NPSA) [49]. Peptides were synthesized according to standard solid-phase synthesis (Sigma-Genosys, The Woodlands, TX). Peptide binding to HLA-A*0201 was analyzed by stabilization of HLA-A*0201 cell surface expression on T2 cells as previously described [17].
Three concentrations (0.5, 5, and 50 mg/ml) were used for each peptide. Stabilization ratios were calculated using mean fluorescence values of (peptide-pulsed T2–
non-pulsed T2)/non-pulsed T2; that way background
HLA-A*0201 expression is set to zero and values above zero reflect binding of peptide.
Preparation of PBMCs From Prostate Cancer Patients and Healthy Volunteers
Blood samples, 20–40 ml per individual, were drawn at one occasion per individual and collected in heparin-coated tubes (BD Biosciences). Peripheral blood mononuclear cells (PBMCs) were obtained by subjecting the samples to Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) density centrifugation.
Samples were from HLA-A2.1þ prostate cancer patients enrolled for radical prostatectomy at the Uppsala University Hospital, Uppsala, Sweden and from healthy age-matched HLA-A2.1þ male and female volunteers. Permission to collect blood samples from prostate cancer patients was approved by the local ethical committee at the Uppsala University Hospital (reference number 01–318). Informed consent was obtained from each patient.
Generation of Peptide-Specif|cTCells
Buffy coats, from approximately 420 ml of periph- eral blood, were obtained from healthy HLA-A2.1þ blood donors at the Uppsala University Hospital Blood Center, Uppsala, Sweden. PBMCs were isolated through Ficoll-Hypaque separation and resuspended in RPMI-1640 supplemented with 10 mM HEPES, 1 mM
L-glutamine, 20 mM 2-mercaptoethanol, 1% pooled normal human serum and 1% PEST. PBMCs were plated in six T-75 culture flasks and after 90 min incubation at 378C, non-adherent peripheral blood lymphocytes (PBLs) were removed and cryopreserved.
Adherent monocytes cultures were either cryopre- served or differentiated into DCs over 8 days as previously described [50]. The mature DC phenotype was confirmed by staining cells with cell surface markers as described above. Mature HLA-A2þ DCs were divided in six portions. Each DC portion was incubated with one prostate antigen peptide (10 mg/ml) for 4 hr and then washed two times with PBS. The peptide-pulsed DCs were pooled and mixed with thawed autologous lymphocytes (non-adherent cell fraction) at a ratio of 1:20. After 12 days autologous monocytes were thawed out, divided in six portions, incubated with the same individual peptides as before being washed and pooled. Lymphocytes were restimu- lated with the peptide-pulsed monocytes as previously described at a ratio of 1:20 [50]. They were restimulated two more times with peptide-pulsed monocytes at weekly intervals before analysis. The peptide pools used were (I) PSA53–61, TGM4612–620, STEAP165–173, STEAP262–270, PAP13–21, hKLK4155–164, (II) TARP(V28L)27–35, TARP(P5L)4–13, PAP13–21, PSMA4–12, PSA53–61, PSA165–174,
(III) hKLK4117–126, NPSA20–28, POTE290–298, STEAP262–270, PSMA4–12, PSMA27–35, (IV) PAP13–22, POTE323–331, AIbZIP70–78, STAMP1402–410, Trp-p8596–605, Trp-p8770–778, (V) PSCA7–15, STAMP1373–382/STEAP232–241, STAMP1402–410, PSA165–174, PAP13–22, PSGR202–210, (VI) STAMP1373–382/STEAP232–241, PSMA27–35, PATE5–13, STEAP165–173, TARP(V28L)27–35, TARP(P5L)4–13. This means that the peptides derived from PSA, PAP, PSMA, TARP, STEAP, and STAMP1 were examined twice whereas the peptides derived from TGM4, PSCA, hKLK4, PSGR, Trp-p8, PATE, POTE, AIbZIP, NPSA were examined once.
Intracellular Interferon Gamma (IFN-g) Staining of TCells
C1R-A2 cells were pulsed with 10 mg/ml of peptides at 378C for 2 hr. They were then washed and incubated at a 1:1 ratio either with PBMCs (from healthy volunteers or prostate cancer patients) or with stimu- lated T cells from healthy volunteers. After 2 hr of incubation at 378C protein secretion was blocked by the addition of 8 mg/ml Brefeldin-A and the incubation was continued for an additional 5 hr. IFN-g production by CD8þT cells was analyzed by intracellular staining and Flow Cytometric analysis as described previously [17,51]. Cell fractions where a distinct population of at least 0.1% of the total CD3þ, CD8þT cell population stained positive for IFN-g were considered positive.
CFSE Proliferation Assay
The CellTrace CFSE cell proliferation kit (Invitrogen, Eugene, OR) was used to evaluate CD8þ T cell proliferation. PBLs were isolated by plastic adhesion of PBMCs obtained from HLA-A2.1þprostate cancer patients and labeled with 10 mM CFSE solution in PBS with 0.1% BSA for 10 min and then washed in fresh media. C1R-A2 cells were pulsed with a peptide pool consisting of STEAP262–270, STAMP1373–382/ STEAP232–241, and STEAP165–173 (10 mg/ml) or with the negative control peptide HIVGag77–85 (SLYNT- VATL) (10 mg/ml) for 2 hr washed in culture media and irradiated (40 Gy). CFSE-labeled PBLs (1 106) were mixed with 2 105 peptide-pulsed C1R-A2 and co- cultured in 12-well plates for 5 days. The cells were incubated with anti-human CD3-APC and CD8-PE antibodies (BD Biosciences) for 30 min at 48C, washed and analyzed by Flow Cytometry.
Statistical Analysis
Statisticon AB, Uppsala, Sweden, performed the statistical analysis. We tested for every peptide the hypothesis of no difference in response (occurrence of
IFN-g-secreting CD8þT cells) between prostate cancer patients and healthy male controls against the hypoth- esis of there being a difference, using Fishers exact test.
A joint P-value for the two groups (prostate cancer patients and healthy male controls) has been derived from the separate P-values by a multiple permutation test with Fisher’s omnibus combination function
2P
i
logðPiÞ. The results for each individual were permuted 10,000 times and the true value of the combination function was compared to the empirical distribution of the 10,000 equivalent values from the permuted datasets. The same analysis was performed to evaluate whether there is a significant difference in occurrence of IFN-g-secreting CD8þ T cells between healthy male and female donors.
RESULTS
Binding of Prostate Antigen-Derived Peptides to HLA-A*0201
The BIMAS and SYFPEITHI online prediction algorithms were utilized to analyze proteins with reported prostate-restricted expression for 9-mer and
10-mer peptides with putative binding to HLA-A*0201.
Based on the results from this analysis we synthesized 24 peptides, derived from 15 proteins, all with BIMAS scores above 78 min and SYFPEITHI scores higher than 20, Table I. Furthermore, all peptides had classical anchor residues, that is, Leucine (L) or Methionine (M) at position 2 and Leucine (L) or Valine (V) at position 9 or 10, respectively. The peptides were evaluated for binding to HLA-A*0201 by incubation with T2 cells at various peptide concentrations. All peptides were able to stabilize HLA-A*0201 expression on the surface of the T2 cells to various degrees, Table I, confirming that they are HLA-A*0201 binders.
C1R-A2 as Peptide-Presenting Cells to Evaluate CD8þTCell Reactivity
The MHC class I expression of C1R-A2 is uniquely HLA-A*0201. We therefore wanted to evaluate whether C1R-A2 can be used as antigen-presenting cells to study HLA-A*0201-restricted peptide-specific CD8þT cell reactivity. A phenotypic characterization of C1R-A2 is presented in Figure 1A,B. It has high HLA- ABC, HLA-DR, CD54, CD80, and CD86 expressions,
TABLE I. Prostate Antigen, Peptide Location, Sequence and HLA-A*0201 Stabilization Ratio onT2 Cells
Prostate-associated antigen Peptide Sequence
BIMAS SYNFP
Stabilization Ratio peptide conc. (mg/ml)
Score Score 0.5 5 50
Prostate-specific antigen PSA53–61 VLVHPQWVL 111 22 0.48 1.30 1.42
PSA165– 174 FLTPKKLQCV 735 26 0.00 0.48 1.07
Prostatic acid phosphatase PAP13– 22 SLSLGFLFLL 1082 26 0.33 1.15 1.15
PAP13– 21 SLSLGFLFL 223 25 0.31 1.05 1.25
Prostate-specific membrane antigen PSMA4 –12 LLHETDSAV 484 25 0.40 1.24 1.50
PSMA27–35 VLAGGFFLL 400 27 0.98 1.57 1.58
Prostate specific transglutaminase TGM4612– 620 TLAIPLTDV 159 27 0.29 0.92 1.39
Prostate stem cell antigen PSCA7 –15 ALLMAGLAL 79 26 0.52 1.22 1.60
TCRg alt. reading frame protein TARP(V28L)27–35 FLFLRNFSL 2108 24 1.14 1.50 1.43 TARP(P5L)4–13 FLPSPLFFFL 5956 21 1.23 1.60 1.35 Six transmembrane epithelial antigen STEAP165–173 GLLSFFFAV 10776 25 0.79 1.20 1.30
prostate STEAP262–270 LLLGTIHAL 309 32 0.79 1.35 1.36
Prostase/human kallikrein 4 hKLK4155–164 LLANGRMPTV 271 27 0.53 1.18 1.36
hKLK4117–126 LMLIKLDESV 154 24 0.36 1.04 1.13
Prostate-spec G protein-coupled receptor PSGR202 –210 ILLVMGVDV 437 26 0.63 1.20 1.44 Prostate-spec protein homolog trp family Trp-p8596 –605 KLLKTLAKV 2071 31 0.00 0.20 0.53 Trp-p8770 –778 VLYSLVFVL 635 27 0.21 0.83 0.62 Prostate and testis expression PATE5 –13 LLLELPILL 550 27 1.32 1.70 1.70 Prostate ovary testis placenta expression POTE323– 331 LLLEQNVDV 1793 28 0.51 1.16 1.23
POTE290– 298 FLIKKKANL 98 26 0.00 0.41 1.03
Six transmembrane protein of prostate 1 STAMP1402 –410 ALLISTFHV 1492 26 0.59 1.20 1.44 STAMP1373 –382/
STEAP232–241
LLAVTSIPSV 271 31 0.99 1.34 1.58
Androgen-induced bZIP AIbZIP70–78 KLFIDPNEV 900 25 0.04 0.37 0.62
Novel prostate-specific antigen NPSA20–28 LLYMRICYV 5534 27 0.07 0.56 0.76
indicating appropriate antigen-presenting properties.
We also compared HLA-A2 surface expression on C1R-A2 with expression on mature monocyte-derived DCs. We found comparable expression levels (Fig. 1B).
Finally, C1R-A2 and mature autologous DCs were evaluated side-by-side as antigen-presenting cells to detect peptide-specific CD8þ T cell in a Flow Cyto- metric-based intracellular IFN-g staining assay. Blood from three healthy donors were analyzed with the STAMP1373–382/STEAP232–241peptide. We found that the percentage of IFN-g-secreting CD3þ, CD8þT cells was 0.3–1.0% when peptide-pulsed C1R-A2 was used and 0.6–1.4% when peptide-pulsed autologous mature DCs was used. One example is shown in Figure 1C.
From these experiments we conclude that C1R-A2 can be used as a universal HLA-A2-restricted peptide- presenting cell line in IFN-g secretion assays with similar results to autologous DCs. This means that one can omit the laborious work of isolating monocytes or DCs from each patient. Furthermore, the stimulator to T cell ratio can easily be controlled and varied when C1R-A2 cells are used. Therefore, C1R-A2 was used in all subsequent IFN-g secretion assays.
Prostate Antigen-Directed CD8þTCells in the Blood of Prostate Cancer Patients
We next analyzed whether CD8þ T cells directed against HLA-A*0201-restricted prostate antigen pep- tides can be found in peripheral blood. Blood samples were collected from 25 HLA-A*0201þprostate cancer patients with localized prostate cancer that were enrolled for radical prostatectomy and from 20 HLA- A*0201þage-matched healthy volunteers (10 males and 10 females). PBLs were isolated and mixed with peptide-pulsed C1R-A2 at a 1:1 ratio for a total of 7 hr (2 hr without blocking and 5 hr with blocked protein secretion). CD8þ T cells were then analyzed for IFN-g production by intracellular IFN-g staining.
We detected circulating antigen-specific T cells in 18
Fig. 1. C1R-A2 is an appropriate stimulator cell line for analysis of peptide-specific IFN-g release from CD8þT cells. A: C1R-A2 was stained with fluorophore-labeled monoclonal antibodies against HLA-ABC, HLA-DR, CD40, CD54, CCR7, CD1a, CD83, CD19, and CD20. B: C1R-A2 and mature DCs were stained with fluorophore- labeled monoclonal antibodies against CD80 and CD86 or with a mouse monoclonal anti-HLA-A2 antibody followed by a fluoro- phore-labeled rabbit anti-mouse antibody. The cells were subse- quently analyzed by Flow Cytometry. Open curves represent staining with specific antibodies to molecules indicated in the top right corner while filled curves represent staining with isotype- relevant negative control antibodies. C: C1R-A2 and mature DCs were incubated with the STAMP1373 ^ 382/STEAP232 ^ 241peptide and used to stimulateT cells (autologous to the DCs).Cells were subse- quently stained with fluorophore-labeled monoclonal anti-CD3, anti-CD8 and anti-IFN-g antibodies and analyzed by Flow Cytome- try. Values presented in the upper right quadrants represent the percentage of IFN-g-secreting T cells within the CD3þ, CD8þ population.One example from threeindependent analysesis shown.
out of 25 prostate cancer patients, that is, we were unable to detect antigen-specific T cells in 7 out of these 25 patients. However, it should be noted that at no instance did we obtain enough patient blood to assay all peptides. One example of T cell analysis from a prostate cancer patient is shown in Figure 2, where 16 peptides were tested and circulating CD8þT cells were detected against six of them, namely TARP27–35, PSMA4–12, PAP13–21, STEAP262–270, STAMP1373–382/STEAP232–241, and hKLK4117–126.
The result from the IFN-g secretion assays in prostate cancer patients and healthy volunteers is presented for each peptide separately in Figure 3. In positive samples
the IFN-g-secreting cells, within the CD3þ, CD8þT cell population, were at least 0.1% and was observed as a distinct population of cells. The frequency of IFN-g- secreting cells was in some cases as high as 2.4%. An irrelevant HLA-A*0201 binding peptide, VMAT131–39, from vesicular monoamine transporter 1 was used as a negative control. It yielded in all cases IFN-g-secreting cells within the CD3þ, CD8þT cell population of less than 0.02% (data not shown). The facts that the negative control peptide did not evoke T cells to produce IFN-g and that no single individual patient presented responses against all peptides prove that the T cell responses observed are peptide-specific and not due to
Fig. 2. An exampleillustrating the detection ofprostate antigen-directed CD8þT cellin theblood of a prostate cancer patient.C1R-A2 cells were separatelypulsedwith HLA-A*0201-bindingpeptides derived fromprostate antigens andmixedwith PBMCs from an HLA-A*0201þpros- tate cancer patient.Cells were subsequently stained with monoclonal fluorophore-labeled anti-CD3, anti-CD8 and anti-IFN-g antibodies and analyzedby Flow Cytometry.Valuespresentedin theupperrightquadrantsrepresentthepercentage ofIFN-g-secretingT cellswithin theCD3þ, CD8þPBMC population.
allogeneic interactions. The number of positive sam- ples out of tested samples for each peptide is presented in Table II. The total numbers of positive samples in the patient group and the healthy male and female groups are presented at the bottom of Table II. Peptide-specific CD8þT cells were detected in 48 out of 184 samples (26.1%) from 25 prostate cancer patients while 17 out of 214 samples (7.9%) were positive in blood from 10 healthy age-matched male volunteers. Fisher’s exact test was used to calculate P-values for each peptide separately, comparing the occurrence of IFN-g-secret- ing CD8þT cells detected in the prostate cancer patient group with the occurrence of IFN-g-secreting CD8þT cells detected in the healthy male donor group. A joint P-value for the two groups was derived from the separate P-values by a multiple permutation test with Fisher’s omnibus combination function to evaluate whether there was a statistical significant difference between prostate cancer patients and healthy male donors. Peptides not yielding T cell responses in
neither of the two groups (PSMA27–35, hKLK4155–164, Trp-p8596–605, POTE290–298, STAMP1402–410, NPSA20–28) were excluded from the statistical analysis. We found that peptide-specific CD8þ T cells were more often detected in the peripheral blood of prostate cancer patients than in healthy age-matched male individuals (a ¼ 0.05; P ¼ 0.0249). Using the same test we found no significant difference in occurrence of IFN-g-secreting CD8þT cells between samples from healthy male and female volunteers (a ¼ 0.05; P ¼ 0.989). Among pep- tides that were sampled more than 10 times, CD8þ T cells were most often found against STEAP262–270
(53%), STAMP1373–382/STEAP232–241(36%), PSA165–174
(30%), TARP27–35 (24%), and TARP4–13 (24%) in the blood of prostate cancer patients, Table II. Furthermore, 15 out of 20 healthy volunteers presented circulating CD8þT cells against STAMP1373–382/STEAP232–241.
In order to investigate peptide-specific T cell proliferation a CFSE assay was set up with PBLs from a prostate cancer patient with very high IFN-g Fig. 3. Prostate antigen-specificCD8þT cellsin theperipheralbloodofprostate cancerpatients andage-matchedhealthyindividuals.PBMCs were isolated from HLA-A*0201þprostate cancer patients (filled circles) and HLA-A*0201þage-matched healthy males (open circles) and females (crosses).They weremixed withpeptide-pulsed C1R-A2 andcells were subsequently stained for CD3,CD8 and IFN-g.Presented values represent the percentage of IFN-g-secreting T cells within the CD3þ, CD8þpopulation after subtracting values obtained from irrelevant peptide-pulsed C1R-A2. Samples with a distinct population of IFN-g-secreting CD8þT cells, representing at least 0.1% of the total CD3þ, CD8þT cell population were consideredpositive.There is substantial overlap of circlesbelow the 0.1% level.
production against the STEAP165–173peptide (14% of CD8þ T cells) (Fig. 4A). CFSE-labeled PBLs were stimulated with peptide-pulsed C1R-A2 (pool of STEAP165–173, STEAP262–270, and STAMP1373–382/ STEAP232–241) and T cell proliferation was measured as dilution of the CFSE dye in each cell division (Fig. 4B). We found that 26.5% of the CD8þ T cells proliferated after STEAP peptide pool stimulation (black line, open curve) as compared to 5.3% of the CD8þT cells after HIV peptide stimulation (gray line, open curve) and 1.2% for unstimulated CD8þT cells (filled curve). We also investigated peptide-specific T cell proliferation using PBLs from prostate cancer patients where between 1% and 2% of CD8þT cells produced IFN-g in response to a prostate antigen peptide. In those cases we did not observe peptide- specific T cell proliferation by using the CFSE assay (data not shown).
Generation of CD8þTCells against Prostate Antigen Peptides
In the cases where healthy volunteers did not have detectable levels of circulating CD8þ T cells against
prostate antigen peptides we investigate the possibility to use an ex vivo stimulation protocol to generate peptide-specific CD8þ T cells. Mature DCs obtained from buffy coat blood of healthy volunteers were divided in six portions and each portion was pulsed with one peptide. The pulsed DCs were pooled and used to stimulate autologous lymphocytes. The lymphocytes were then restimulated three times at weekly intervals with peptide-pulsed monocytes.
Thereafter, T cells were analyzed for specific IFN-g production using peptide-pulsed C1R-A2 as stimula- tors. By using this ex vivo activation approach we were able to generate IFN-g-secreting CD8þ T cells against TGM4612–620, STEAP165–173, and PSA53–61
simultaneously from one blood donor (I), TARP27–35
and TARP4–13from another (II), and hKLK4117–126and NPSA20–28from a third donor (III) (Fig. 5). We were also able to generate CD8þT cells that specifically produced IFN-g against PAP13–22 (IV), PSCA7–15 (V), and STAMP1373–382/STEAP232–241 (VI) with frequencies displayed in Figure 5. When stimulated T cells were assayed against C1R-A2 cells pulsed with an irrelevant HLA-A*0201-binding peptide, not used during stim- TABLE II. Prostate Antigen-Reactive CD8þT Cells in the Peripheral Blood of Prostate Cancer Patients and Healthy Age- Matched Male and Female Individuals
Peptide Prostate cancer patients Healthy male donors Healthy female donors
PSA53–61 3/13 23% 0/10 0% 0/10 0%
PSA165–174 6/20 30% 0/10 0% 0/10 0%
PAP13–22 1/5 20% 0/9 0% 0/10 0%
PAP13–21 2/11 18% 0/9 0% 0/10 0%
PSMA4–12 3/16 19% 1/10 10% 0/10 0%
PSMA27–35 0/1 0% 0/9 0% 0/10 0%
TGM4612–620 0/1 0% 2/10 20% 2/10 20%
PSCA7– 15 2/10 20% 1/4 25% 0/3 0%
TARP27–35 5/21 24% 1/10 10% 1/10 10%
TARP4 –13 5/21 24% 0/10 0% 0/10 0%
STEAP165– 173 1/7 14% 1/4 25% 0/3 0%
STEAP262– 270 8/15 53% 0/10 0% 0/10 0%
hKLK4155–164 0/1 0% 0/10 0% 0/10 0%
hKLK4117–126 3/9 33% 1/10 10% 1/10 10%
PSGR202– 210 2/4 50% 1/10 10% 0/10 0%
Trp-p8596– 605 0/1 0% 0/10 0% 0/10 0%
Trp-p8770– 778 0/1 0% 1/6 17% 1/7 14%
PATE5– 13 0/3 0% 1/10 10% 0/10 0%
POTE323–331 1/3 33% 0/10 0% 0/10 0%
POTE290–298 0/1 0% 0/9 0% 0/10 0%
STAMP1402– 410 0/1 0% 0/10 0% 0/10 0%
STAMP1373– 382/STEAP232– 241 5/14 36% 7/10 70% 8/10 80%
AibZIP70–78 1/3 33% 0/10 0% 0/10 0%
NPSA20– 28 0/2 0% 0/4 0% 0/1 0%
Total frequency 26.1% (48/184) 7.9% (17/214) 6.1% (13/214)
Positives are between 0.1% and 2.4% of CD8þT cells.
ulation, the frequencies of IFN-g-secreting CD8þT cells were always less than or equal to 0.02% (Fig. 5).
DISCUSSION
HLA-A*0201 is the most commonly expressed HLA allele in the Caucasian, African American and Latin American populations and the second most commonly expressed HLA allele, after HLA-A*1101, in the Asian American population [52]. Over the last decade considerable efforts have been carried out to develop prostate cancer vaccines by using HLA-A*0201- restricted peptides derived from antigens associated
with prostate cancer cells [9–21,26]. In almost all instances single peptides were used to illustrate a proof of principle. However, in order to obtain a polyclonal and long-lasting T cell response against prostate cancer cells it will be of importance to use several peptides from numerous prostate antigens Fig. 4. IFN-g-producing STEAP-specific CD8þT cells proliferate
upon stimulation with a pool of STEAP peptides. A: C1R-A2 was incubated with STEAP262 ^ 270, STAMP1373 ^ 382/STEAP232 ^ 241 or STEAP165 ^173peptides and used to stimulate PBLs from a prostate cancer patient. Cells were subsequently stained with fluorophore- labeled monoclonal anti-CD3, anti-CD8 and anti-IFN-g antibodies andanalyzedby Flow Cytometry.Valuespresentedin theupperright quadrantsrepresentthepercentage ofIFN-g-secretingT cellswithin the CD3þ, CD8þpopulation. B: PBLs from the same patient were labeled with CFSE and stimulated with peptide-pulsed C1R-A2.
After five days, cells were stained with fluorophore-labeled mono- clonal anti-CD3 and anti-CD8 antibodies and analyzed by Flow Cytometry. Filled curve illustrates proliferation of unstimulated CD3þ,CD8þT cells.Black line (open curve) illustrates proliferation of CD3þ,CD8þT cells stimulated with STEAP peptide pool-pulsed C1R-A2 cells.Gray line (open curve) illustrates background prolifer- ation of CD3þ, CD8þT cells stimulated with HIV peptide-pulsed C1R-A2 cells.
Fig. 5. CD8þT cells generated against HLA-A*0201-binding pep- tides from prostate antigens. Peripheral blood samples from six healthy individuals (I, II, III, IV, V, and VI) without detectable levels of circulating CD8þT cells against any of the prostate antigens were used. PBLs were stimulated once with a pool of peptide- pulsed autologous mature DCs and re-stimulated three times with pooled peptide-pulsed autologous monocytes. The peptide pools used for the six blood samples are described in the Materials and Methods Section. The stimulated T cells were then analyzed for IFN-g secretion in response to encounter with peptide-pulsed C1R-A2. Cells were stained with monoclonal fluorophore-labeled anti-CD3, anti-CD8, and anti-IFN-g antibodies and subsequently analyzed by Flow Cytometry. Values presented in the upper right quadrantsrepresent thepercentage ofIFN-g-secretingTcellswithin the CD3þ,CD8þpopulation.
simultaneously. This would minimize the risk of obtaining antigen-loss tumor variants in vivo, as has for example been reported in a melanoma immuno- therapy study [53]. Furthermore, close attention must be paid on the potential tolerogenic state of prostate cancer, in particular the suppressive role of regulatory T cells [54]. It has recently been shown that CD4þCD25highT cells are enriched in the tumor and peripheral blood of prostate cancer patients [55]. Such cells may have to be repressed in conjunction with the adoptive transfer of antigen-specific CTLs.
We show herein that prostate cancer patients diag- nosed with localized disease frequently have detectable levels of CD8þT cells against self antigen expressed by prostate cancer cells, indicating that the immune system is trying to mount a T cell response against the tumors.
Prostate antigen-specific CD8þT cells were more often found in the peripheral blood of prostate cancer patients enrolled for radical prostatectomy than in healthy age- matched individuals. The existence of circulating prostate antigen-specific CD8þ T cells was also con- firmed by a proliferation assay using blood from a prostate cancer patient with high frequencies of IFN-g- producing CD8þT cells against a STEAP epitope. No significant difference was observed between blood samples from healthy male and female individuals with regards to circulating prostate antigen-specific CD8þ T cells, indicating that there is no general tendency in healthy males to have an increased level of T cells directed against ‘‘male’’ prostate antigens.
Interestingly, 15 out of 20 HLA-A*0201þ healthy volunteers (both males and females) present circulating CD8þT cells against the STAMP1373–382/STEAP232–241
peptide (LLAVTSIPSV). The reason for this is not yet known, but it indicates that STAMP1 and/or STEAP are immunogenic proteins that should be further evaluated for their potential role as auto-antigens.
Furthermore, there is a tendency that the occurrence of circulating STAMP1373–382/STEAP232–241-specific CD8þT cells is less frequent in prostate cancer patients than in healthy age-matched individuals.
It has been reported in the past that prostate cancer patients may have circulating CD8þ T cells directed against HLA-A*0201-restricted peptides derived from prostate antigens. For example, T cells that produce IFN-g in response to PSA165–174[56] (in one reference referred to as PSA141–150 [57]), PSCA99–107 [20], PSCA105–113[20], TARP27–35[18] and STEAP86–94[23]
have been reported. We now expand the list by adding a number of new HLA-A*0201-restricted peptides from prostate antigen against which circulating CD8þT cells can be detected in prostate cancer patients, namely PSA53–61, PAP13–21,22, PSMA4–12, PSCA7–15, TARP4–12, STEAP165–173, STEAP262–270, hKLK4117–126, PSGR202–210, POTE323–331, STAMP1373–382, and AibZIP20–28. The
plethora of HLA-A*0201-restricted peptides available from prostate antigens makes prostate cancer an interesting disease for adoptive T cell therapy.
We also investigated the frequency of IFN-g-pro- ducing T cells in late stage prostate cancer patients using the HLA-A*0201-restricted peptides derived from PSA, TARP, and STEAP. We obtained PBLs from 10 HLA-A*0201þ prostate cancer patients with bone and/or lymph node metastases. Nine out of 10 patients were on hormonal treatment at the time of blood collection and 8 out of 10 patients had obtained palliative irradiation treatment of meta- stases in the past. A general observation was that the PBL counts were low. Furthermore, we did not detect IFN-g-producing CD8þ T cells in response to in vitro peptide stimulation for any of the 10 patients (data not shown). This indicates that the CTL immune response against prostate cell-associated antigens is low or non-existing in late stage prostate cancer patients and that T cell-based immunotherapy may have higher chance of being successful if applied at an early stage.
In the cases where circulating CD8þT cells were not detected we show that it is possible to break tolerance by generating CD8þT cells that secreted IFN-g against 10 of the 25 evaluated peptides. We describe a protocol where DCs/monocytes are divided into six groups before being pulsed with individual peptides, in order to avoid binding competition among peptides. The peptide-pulsed cells are then pooled and used for autologous lymphocyte stimulations. Importantly, by using this protocol we were able to generate T cells against multiple prostate antigens simultaneously from a naı¨ve T cell repertoire. It may therefore overcome the potential problem of the development of spontaneous antigen-loss tumor variants in vivo. We made four stimulations before assessing T cell reac- tivity and did not investigate whether peptide specific- ity could be observed already after one, two or three stimulations. Repeated T cell stimulation can result in low avidity T cells that may be unable to recognize cells that endogenously express and present the antigen.
However, with an appropriately designed stimula- tion/restimulation protocol high-avidity T cells can repeatedly be obtained [58]. To our knowledge there is no prostate cancer cell line that efficiently express HLA-A*0201 on the cell surface and at the same time endogenously express multiple prostate TAAs [17,59].
Therefore, the avidity of the T cells obtained in our protocol is currently not known.
In conclusion, the fact that CD8þ T cells directed against prostate antigen peptides can be found in, or generated from peripheral blood strongly suggests that such T cells could be expanded ex vivo for adoptive transfer to prostate cancer patients.
ACKNOWLEDGMENTS
The authors like to thank Jimmy Larsson for help with the peptide-binding analysis. The Swedish Cancer Society (Grant 4419-B06-07XCC), Gunnar Nilsson’s Cancer Foundation (E35/07) and the Lion’s Cancer Foundation in Uppsala supported the work. ME is a recipient of the Go¨ran Gustafsson’s Award.
REFERENCES
1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Smigal C, Thun MJ.
Cancer statistics, 2006. CA Cancer J Clin 2006;56(2):106–130.
2. Rosenberg SA. Progress in human tumour immunology and immunotherapy. Nature 2001;411(6835):380–384.
3. Pavlenko M, Roos AK, Lundqvist A, Palmborg A, Miller AM, Ozenci V, Bergman B, Egevad L, Hellstrom M, Kiessling R, Masucci G, Wersall P, Nilsson S, Pisa P. A phase I trial of DNA vaccination with a plasmid expressing prostate-specific antigen in patients with hormone-refractory prostate cancer. Br J Cancer 2004;91(4):688–694.
4. Murphy G, Tjoa B, Ragde H, Kenny G, Boynton A. Phase I clinical trial: T-cell therapy for prostate cancer using autologous dendritic cells pulsed with HLA-A0201-specific peptides from prostate- specific membrane antigen. Prostate 1996;29(6):371–380.
5. Tjoa BA, Simmons SJ, Bowes VA, Ragde H, Rogers M, Elgamal A, Kenny GM, Cobb OE, Ireton RC, Troychak MJ, Salgaller ML, Boynton AL, Murphy GP. Evaluation of phase I/II clinical trials in prostate cancer with dendritic cells and PSMA peptides.
Prostate 1998;36(1):39–44.
6. Gattinoni L, Powell DJ, Jr., Rosenberg SA, Restifo NP. Adoptive immunotherapy for cancer: Building on success. Nat Rev Immunol 2006;6(5):383–393.
7. Blattman JN, Greenberg PD. Cancer immunotherapy: A treat- ment for the masses. Science 2004;305(5681):200–205.
8. Essand M. Gene therapy and immunotherapy of prostate cancer:
Adenoviral-based strategies. Acta Oncol 2005;44(6):610–627.
9. Heiser A, Dahm P, Yancey DR, Maurice MA, Boczkowski D, Nair SK, Gilboa E, Vieweg J. Human dendritic cells transfected with RNA encoding prostate-specific antigen stimulate prostate- specific CTL responses in vitro. J Immunol 2000;164(10):5508–
5514.
10. Correale P, Walmsley K, Nieroda C, Zaremba S, Zhu M, Schlom J, Tsang KY. In vitro generation of human cytotoxic T lymphocytes specific for peptides derived from prostate-specific antigen. J Natl Cancer Inst 1997;89(4):293–300.
11. Tjoa B, Boynton A, Kenny G, Ragde H, Misrock SL, Murphy G.
Presentation of prostate tumor antigens by dendritic cells stimulates T-cell proliferation and cytotoxicity. Prostate 1996;28(1):65–69.
12. Lu J, Celis E. Recognition of prostate tumor cells by cytotoxic T lymphocytes specific for prostate-specific membrane antigen.
Cancer Res 2002;62(20):5807–5812.
13. Peshwa MV, Shi JD, Ruegg C, Laus R, van Schooten WC.
Induction of prostate tumor-specific CD8þ cytotoxic T-lympho- cytes in vitro using antigen-presenting cells pulsed with prostatic acid phosphatase peptide. Prostate 1998;36(2):129–
138.
14. Dannull J, Diener PA, Prikler L, Furstenberger G, Cerny T, Schmid U, Ackermann DK, Groettrup M. Prostate stem cell antigen is a promising candidate for immunotherapy of advanced prostate cancer. Cancer Res 2000;60(19):5522–5528.
15. Francini G, Scardino A, Kosmatopoulos K, Lemonnier FA, Campoccia G, Sabatino M, Pozzessere D, Petrioli R, Lozzi L, Neri P, Fanetti G, Cusi MG, Correale P. High-affinity HLA-A(*)02.01 peptides from parathyroid hormone-related protein generate in vitro and in vivo antitumor CTL response without auto- immune side effects. J Immunol 2002;169(9):4840–4849.
16. Meidenbauer N, Harris DT, Spitler LE, Whiteside TL. Gener- ation of PSA-reactive effector cells after vaccination with a PSA- based vaccine in patients with prostate cancer. Prostate 2000;
43(2):88–100.
17. Carlsson B, Totterman TH, Essand M. Generation of cytotoxic T lymphocytes specific for the prostate and breast tissue antigen TARP. Prostate 2004;61(2):161–170.
18. Oh S, Terabe M, Pendleton CD, Bhattacharyya A, Bera TK, Epel M, Reiter Y, Phillips J, Linehan WM, Kasten-Sportes C, Pastan I, Berzofsky JA. Human CTLs to wild-type and enhanced epitopes of a novel prostate and breast tumor-associated protein, TARP, lyse human breast cancer cells. Cancer Res 2004;64(7):2610–2618.
19. Kiessling A, Fussel S, Schmitz M, Stevanovic S, Meye A, Weigle B, Klenk U, Wirth MP, Rieber EP. Identification of an HLA- A*0201-restricted T-cell epitope derived from the prostate cancer-associated protein trp-p8. Prostate 2003;56(4):270–279.
20. Kiessling A, Schmitz M, Stevanovic S, Weigle B, Holig K, Fussel M, Fussel S, Meye A, Wirth MP, Rieber EP. Prostate stem cell antigen: Identification of immunogenic peptides and assessment of reactive CD8þ T cells in prostate cancer patients. Int J Cancer 2002;102(4):390–397.
21. Kiessling A, Stevanovic S, Fussel S, Weigle B, Rieger MA, Temme A, Rieber EP, Schmitz M. Identification of an HLA-A*0201- restricted T-cell epitope derived from the prostate cancer- associated protein prostein. Br J Cancer 2004;90(5):1034–1040.
22. Friedman RS, Spies AG, Kalos M. Identification of naturally processed CD8 T cell epitopes from prostein, a prostate tissue- specific vaccine candidate. Eur J Immunol 2004;34(4):1091–1101.
23. Alves PM, Faure O, Graff-Dubois S, Cornet S, Bolonakis I, Gross DA, Miconnet I, Chouaib S, Fizazi K, Soria JC, Lemonnier FA, Kosmatopoulos K. STEAP, a prostate tumor antigen, is a target of human CD8(þ) T cells. Cancer Immunol Immunother 2006.
24. Machlenkin A, Paz A, Bar Haim E, Goldberger O, Finkel E, Tirosh B, Volovitz I, Vadai E, Lugassy G, Cytron S, Lemonnier F, Tzehoval E, Eisenbach L. Human CTL epitopes prostatic acid phosphatase-3 and six-transmembrane epithelial antigen of prostate-3 as candidates for prostate cancer immunotherapy.
Cancer Res 2005;65(14):6435–6442.
25. Rodeberg DA, Nuss RA, Elsawa SF, Celis E. Recognition of six- transmembrane epithelial antigen of the prostate-expressing tumor cells by peptide antigen-induced cytotoxic T lympho- cytes. Clin Cancer Res 2005;11(12):4545–4552.
26. Marrari A, Iero M, Pilla L, Villa S, Salvioni R, Valdagni R, Parmiani G, Rivoltini L. Vaccination therapy in prostate cancer.
Cancer Immunol Immunother 2007;56(4):429–445.
27. Yokokawa J, Bera TK, Palena C, Cereda V, Remondo C, Gulley JL, Arlen PM, Pastan I, Schlom J, Tsang KY. Identification of cytotoxic T-lymphocyte epitope(s) and its agonist epitope(s) of a novel target for vaccine therapy (PAGE4). Int J Cancer 2007;121(3):595–605.
28. Edwards PA, Smith CM, Neville AM, O’Hare MJ. A human- hybridoma system based on a fast-growing mutant of the ARH- 77 plasma cell leukemia-derived line. Eur J Immunol 1982;
12(8):641–648.
29. Storkus WJ, Howell DN, Salter RD, Dawson JR, Cresswell P. NK susceptibility varies inversely with target cell class I HLA antigen expression. J Immunol 1987;138(6):1657–1659.
30. Hogan KT, Shimojo N, Walk SF, Engelhard VH, Maloy WL, Coligan JE, Biddison WE. Mutations in the alpha 2 helix of HLA- A2 affect presentation but do not inhibit binding of influenza virus matrix peptide. J Exp Med 1988;168(2):725–736.
31. Anderson KS, Alexander J, Wei M, Cresswell P. Intracellular transport of class I MHC molecules in antigen processing mutant cell lines. J Immunol 1993;151(7):3407–3419.
32. Parker KC, Bednarek MA, Coligan JE. Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. J Immunol 1994;
152(1):163–175.
33. Rammensee H, Bachmann J, Emmerich NP, Bachor OA, Stevanovic S. SYFPEITHI: Database for MHC ligands and peptide motifs. Immunogenetics 1999;50(3–4):213–219.
34. Lundwall A, Lilja H. Molecular cloning of human prostate specific antigen cDNA. FEBS Lett 1987;214(2):317–322.
35. Yeh LC, Lee AJ, Lee NE, Lam KW, Lee JC. Molecular cloning of cDNA for human prostatic acid phosphatase. Gene 1987;60 (2–3):191–196.
36. Israeli RS, Powell CT, Corr JG, Fair WR, Heston WD. Expression of the prostate-specific membrane antigen. Cancer Res 1994;
54(7):1807–1811.
37. Grant FJ, Taylor DA, Sheppard PO, Mathewes SL, Lint W, Vanaja E, Bishop PD, O’Hara PJ. Molecular cloning and characterization of a novel transglutaminase cDNA from a human prostate cDNA library. Biochem Biophys Res Commun 1994;203(2):1117–1123.
38. Reiter RE, Gu Z, Watabe T, Thomas G, Szigeti K, Davis E, Wahl M, Nisitani S, Yamashiro J, Le Beau MM, Loda M, Witte ON.
Prostate stem cell antigen: A cell surface marker overexpressed in prostate cancer. Proc Natl Acad Sci USA 1998;95(4):1735–
1740.
39. Essand M, Vasmatzis G, Brinkmann U, Duray P, Lee B, Pastan I.
High expression of a specific T-cell receptor gamma transcript in epithelial cells of the prostate. Proc Natl Acad Sci USA 1999;
96(16):9287–9292.
40. Wolfgang CD, Essand M, Vincent JJ, Lee B, Pastan I. TARP: A nuclear protein expressed in prostate and breast cancer cells derived from an alternate reading frame of the T cell receptor gamma chain locus. Proc Natl Acad Sci USA 2000;97(17):9437–9442.
41. Hubert RS, Vivanco I, Chen E, Rastegar S, Leong K, Mitchell SC, Madraswala R, Zhou Y, Kuo J, Raitano AB, Jakobovits A, Saffran DC, Afar DE. STEAP: A prostate-specific cell-surface antigen highly expressed in human prostate tumors. Proc Natl Acad Sci USA 1999;96(25):14523–14528.
42. Nelson PS, Gan L, Ferguson C, Moss P, Gelinas R, Hood L, Wang K. Molecular cloning and characterization of prostase, an androgen-regulated serine protease with prostate-restricted expression. Proc Natl Acad Sci USA 1999;96(6):3114–3119.
43. Xu LL, Stackhouse BG, Florence K, Zhang W, Shanmugam N, Sesterhenn IA, Zou Z, Srikantan V, Augustus M, Roschke V, Carter K, McLeod DG, Moul JW, Soppett D, Srivastava S. PSGR, a novel prostate-specific gene with homology to a G protein- coupled receptor, is overexpressed in prostate cancer. Cancer Res 2000;60(23):6568–6572.
44. Tsavaler L, Shapero MH, Morkowski S, Laus R. Trp-p8, a novel prostate-specific gene, is up-regulated in prostate cancer and other malignancies and shares high homology with transient receptor potential calcium channel proteins. Cancer Res 2001;
61(9):3760–3769.
45. Bera TK, Maitra R, Iavarone C, Salvatore G, Kumar V, Vincent JJ, Sathyanarayana BK, Duray P, Lee BK, Pastan I. PATE, a gene expressed in prostate cancer, normal prostate, and testis,
identified by a functional genomic approach. Proc Natl Acad Sci USA 2002;99(5):3058–3063.
46. Bera TK, Zimonjic DB, Popescu NC, Sathyanarayana BK, Kumar V, Lee B, Pastan I. POTE, a highly homologous gene family located on numerous chromosomes and expressed in prostate, ovary, testis, placenta, and prostate cancer. Proc Natl Acad Sci USA 2002;99(26):16975–16980.
47. Korkmaz KS, Elbi C, Korkmaz CG, Loda M, Hager GL, Saatcioglu F. Molecular cloning and characterization of STAMP1, a highly prostate-specific six transmembrane protein that is overexpressed in prostate cancer. J Biol Chem 2002;
277(39):36689–36696.
48. Qi H, Fillion C, Labrie Y, Grenier J, Fournier A, Berger L, El-Alfy M, Labrie C. AIbZIP, a novel bZIP gene located on chromosome 1q21.3 that is highly expressed in prostate tumors and of which the expression is up-regulated by androgens in LNCaP human prostate cancer cells. Cancer Res 2002;62(3):721–733.
49. Naz RK, Santhanam R, Tyagi N. Novel human prostate-specific cDNA: Molecular cloning, expression, and immunobiology of the recombinant protein. Biochem Biophys Res Commun 2002;
297(5):1075–1084.
50. Carlsson B, Cheng WS, Totterman TH, Essand M. Ex vivo stimulation of cytomegalovirus (CMV)-specific T cells using CMV pp65-modified dendritic cells as stimulators. Br J Haematol 2003;121(3):428–438.
51. Nomura LE, Walker JM, Maecker HT. Optimization of whole blood antigen-specific cytokine assays for CD4(þ) T cells.
Cytometry 2000;40(1):60–68.
52. Cao K, Hollenbach J, Shi X, Shi W, Chopek M, Fernandez-Vina MA. Analysis of the frequencies of HLA-A, B, and C alleles and haplotypes in the five major ethnic groups of the United States reveals high levels of diversity in these loci and contrasting distribution patterns in these populations. Hum Immunol 2001;62(9):1009–1030.
53. Yee C, Thompson JA, Byrd D, Riddell SR, Roche P, Celis E, Greenberg PD. Adoptive T cell therapy using antigen-specific CD8þ T cell clones for the treatment of patients with metastatic melanoma: In vivo persistence, migration, and antitumor effect of transferred T cells. Proc Natl Acad Sci USA 2002;99(25):16168–
16173.
54. Zou W. Regulatory T cells, tumour immunity and immunother- apy. Nat Rev Immunol 2006;6(4):295–307.
55. Miller AM, Lundberg K, Ozenci V, Banham AH, Hellstrom M, Egevad L, Pisa P. CD4þCD25high T cells are enriched in the tumor and peripheral blood of prostate cancer patients.
J Immunol 2006;177(10):7398–7405.
56. Chakraborty NG, Stevens RL, Mehrotra S, Laska E, Taxel P, Sporn JR, Schauer P, Albertsen PC. Recognition of PSA-derived peptide antigens by T cells from prostate cancer patients without any prior stimulation. Cancer Immunol Immunother 2003;
52(8):497–505.
57. Alexander RB, Brady F, Leffell MS, Tsai V, Celis E. Specific T cell recognition of peptides derived from prostate-specific antigen in patients with prostate cancer. Urology 1998;51(1):150–157.
58. Ho WY, Nguyen HN, Wolfl M, Kuball J, Greenberg PD. In vitro methods for generating CD8þ T-cell clones for immunotherapy from the naive repertoire. J Immunol Methods 2006;310(1–2):
40–52.
59. Carlsson B, Forsberg O, Bengtsson M, Totterman TH, Essand M.
Characterization of human prostate and breast cancer cell lines for experimental T cell-based immunotherapy. Prostate 2007;
67(4):389–395.
